1.1 Field of the Invention
The present invention relates to a genetic association between interleukins and low birth weight. Particularly, the invention relates to using fetal tissue to predict low birth weight delivery. The invention also provided kits for determination of susceptibility to low birth weight deliveries.
1.2 Brief Description of the Prior Art
In the United States there are about 250,000 spontaneous preterm births each year, occurring at less than 37 weeks gestation, resulting in birth weights under 2500 g or 5xc2xd pounds. This obstetric problem accounts for about two-thirds of all neonatal mortality and over 5 billion dollars in ICU hospital costs each year. Furthermore, permanent disability among survivors is high, especially for respiratory and neurological disorders. Despite increased attention to maternal prenatal care over the last forty years and advances in medical care, most of the advances in infant mortality have been gained through improved capacity to improve the survival of the very low birth weight infants (i.e., those infants having a birth weight of  less than 1500 g). Unfortunately, the incidence of spontaneous preterm births (SPB) has not significantly changed over the last decade. This has been attributed to the recognition that there is currently little understanding of the antecedent risk factors associated with spontaneous preterm births. Furthermore, there are currently no tests for identify mothers or fetuses at high risk. Recent data suggest that as much as 60% of the observed SPB has no suspected etiology. Maternal systemic infections and especially bacterial vaginosis have been shown to be an important source of preterm deliveries and histologic chorioamnionitis, which is highly correlated with SPB. However, other infectious or inflammatory processes may also be involved in SPB. Especially, since intraamniotic increases in PGE2, IL-1 and TNF are consistently found in SPB, even in the absence of detectable infection.
Bacterial infections are associated with premature low birth weight (PLBW). Bacterial infections in the genitourinary tract have been reported to be a major risk factor for preterm delivery (1-4). In the largest study conducted to date by Hillier et al. (2) the relationship between bacterial vaginosis, preterm delivery and low birth weight was explored. In the study Hillier and her colleagues followed more than 10,000 women, from seven medical centers, from 23-26 weeks gestation through delivery. Vaginal cultures were taken to ascertain if bacterial vaginosis was present. Of the 10,397 women, 4.8% delivered PLBW infants. During this study it was also suggested that women who had urinary tract infections or used antibiotics prior to enrollment in the study were also more likely to deliver a PLBW infant. The data from this study demonstrated that 16% of the study population had bacterial vaginosis (BV) and that those 16% were 40% more likely to give birth prematurely than women without BV. The study also determined that bacterial vaginosis was associated with the preterm delivery of low birth weight infants independently of other recognized risk factors.
In other studies conducted by McGregor et al. (4) and Gravett et al. (5) it was postulated that the presence of bacterial vaginosis is associated with subclinical amniotic fluid infection in women with intact fetal membranes; an increased risk of abortion at less than 22 weeks; premature rupture of membranes (PROM), and preterm birth. Other studies have also shown the association between bacterial vaginosis and amniotic fluid infection, histological and clinical chorioamnionitis, placental infection, PROM, premature labor, preterm delivery and a higher maternal infectious morbidity postpartum (5-20). Thus, it appears likely that the genitourinary tract represents a major source of potential infectious challenge that contributes to PLBW.
Recently, a more distant chronic bacterial infection in the oral cavity, periodontitis, has, in certain aspects, been associated with PLBW deliveries. Offenbacher et al. (21) conducted a case control study on 124 pregnant or post-partum women. PLBW cases were defined as a mother with a birth weight of less than 2500 grams and one or more of the following: gestational age  less than 37 weeks, preterm labor (PTL), or preterm premature rupture of membranes (PPROM). Controls were all normal birth weight infants (NBW). Certain types of severe periodontal disease was associated with an increased risk of PLBW (adjusted odds ratio of 7) after controlling for known obstetric PLBW risk factors such as smoking, race, alcohol usage, age, nutrition and genitourinary tract infection.
The primary bacteria involved in the genitourinary and periodontal infections are Gram-negative and are known to release the endotoxin lipopolysaccharide (LPS) into the tissue environment. There is substantial evidence that LPS is associated with pregnancy complications in animals. Endotoxins from enteric bacteria are capable of inducing placental necrosis, spontaneous abortions, fetal organ damage, fetal death and malformations (22).
When challenged with E. coli LPS, Lanning et al. (23) found that the embryological development of the golden hamster was affected, resulting in malformations, spontaneous abortions and low fetal weight. These series of experiments clearly demonstrated that infections in pregnant animals could elicit many pregnancy complications including spontaneous abortion, preterm labor, low birth weight, fetal growth restriction and skeletal anomalies. These experiments also supported the hypothesis that the bacteria associated with bacterial vaginosis, pelvic inflammatory disease and other sexually transmitted diseases have the potential to induce alterations that become evident at the outcomes of pregnancy.
In addition, recent studies (24, 25) involving the bacteria that are involved in periodontitis further suggest that chronic, non-disseminating infections, including those at distant sites, may strongly influence fetal outcomes. Of critical importance to these early experiments was the demonstration that these low-grade infections with low numbers of oral pathogens were not of sufficient magnitude to induce maternal malaise or fever. There was, however, a measurable local increase in PGE and TNFxcex1, as well as a 15-18% decrease in fetal weight (26). Furthermore, the magnitude of the PGE2 and TNFxcex1 response was inversely related to the weight of the fetuses, mimicking the intraamniotic changes seen in humans with PLBW (26). LPS dosing experiments demonstrated that higher levels of LPS could induce fever and weight loss in pregnant animals and resulted in more severe pregnancy outcomes including spontaneous abortions and malformations. These more dramatic outcomes were not seen in the low challenge-oral infection models, but rather resulted in a consistent decrease in fetal weight, and previous sensitizations or exposures to these pathogens prior to pregnancy enhanced the severity of the fetal growth restriction when a secondary exposure occurred during pregnancy (24, 25).
Inflammatory mediators such as prostaglandin E2 (PGE2) and interleukin-1 (IL-1) are present not only in all immuno-inflammatory processes, but also regulate the normal physiologic process of parturition, as well as pathologic prematurity. Amniotic fluid levels of PGE2 rise steadily throughout pregnancy until a critical threshold level is reached to induce labor, cervical dilation and delivery.
The role of prostaglandins in regulating the normal physiology of pregnancy has been well documented. Gibbs et al. (27) summarized the evidence supporting the role of prostaglandins in human labor. Treatment with prostaglandin inhibitors delays the process of mid-trimester abortion and the onset of labor and can arrest preterm labor. Parturition at term is associated with elevated amniotic fluid and maternal plasma concentrations of prostaglandins.
The association between preterm labor and changes in amniotic fluid concentrations of PGE2 and prostaglandin F2xcex1 (PGF2xcex1) was studied in 30 women by Mazor et al. (28). They demonstrated that women with preterm labor and intraamniotic infection had significantly higher amniotic fluid concentrations of PGE2 and PGF2xcex1 than women with preterm labor but without infection. This may be explained by the fact that amnion from women with preterm labor and histologic chorioamnionitis produced more PGE2 than amnion from women without placental inflammation (29). Romero and co-workers (30) observed elevated levels of PGE2 as a consistent and reproducible fact of PLBW even in the absence of clinical or subclinical genitourinary tract infection and they postulated that the majority of PLBW deliveries are xe2x80x9cprobably caused by an infection of unknown origin.xe2x80x9d
Tamatani has shown that interleukin-1 beta (IL-1xcex2) is present in normal amniotic fluid (31) and Flynn has demonstrated production of IL-1xcex2 by human placental macrophages (32). The small amount of IL-1xcex2 detected in the second trimester amniotic fluid has been shown to exhibit a threefold increase with the onset of labor (33). Kent et al. (34) in a study on the effects of IL-1xcex2 on prostaglandin production by cultured human fetal membranes, has demonstrated that IL-1xcex2 is a potent stimulator of the synthesis of prostaglandins by decidua and by amnion. IL-1 was the first cytokine implicated in the onset of labor in the presence of infection. IL-1 is produced in vitro by human decidua in response to bacterial products (35,36). In patients with preterm labor and bacteria in the amniotic cavity, amniotic fluid IL-1 bioactivity and concentrations are elevated (36). Placental necrosis and fetal resorption can be induced in rats by the injection of recombinant human IL-1xcex2 on day 12 of gestation (37). Romero et al. (36) have also demonstrated among patients with PROM and bacteria in the amniotic cavity, that amniotic fluid IL-1xcex2 bioactivity and concentration is elevated with labor compared to those without labor. IL-1xcex2 stimulates prostaglandin production by amnion and decidua in vitro (38).
The role of infection and inflammatory mediators in pregnant hamsters has also been examined (39). These animals were infected in the oral cavity with bacteria known to cause periodontitis. After periodontitis was established in the hamsters, a statistically significant elevation of intraamniotic fluid levels of both PGE, and TNFxcex1 was observed, providing direct evidence that infections throughout the body can result in a change in the fatal environment. It is possible that both PGE and TNFxcex1 are produced by the periodontium and appear in the systemic circulation to eventually cross the chorioamniotic barrier and finally appear in the fluid. However, it seems more likely that blood borne bacterial products, especially LPS, target the chorioamniotic plexus to trigger local PGE and TNFxcex1 synthesis. Irrespective of targeting mechanism, it is clear that experimental infections in animal model can provide sufficient challenge to elicit LBW.
The data described above have led to the current clinical concept of the cause of PLBW outcomes, which may be described by the statements that are shown diagrammatically in FIGS. 1 and 2. Maternal infections activates the maternal inflammatory response which can then lead to an adverse pregnancy outcome (FIG. 2). Additionally, a maternal infection may activate the fetal inflammatory response, which in turn leads to an adverse pregnancy outcome (FIG. 1).
In the last few years, the more severe forms of several diseases were shown to be associated with genetic variations in the genes for the inflammatory cytokines IL-1xcex1, IL-1xcex2, IL-1 receptor antagonist (IL-1RA or IL-1RN), and TNFxcex1. These findings, together with new insights about PLBW, has led to a new hypothesis of how infections and inflammatory mediators influence pregnancy outcomes. This is shown diagrammatically in FIG. 3.
The cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF) are important mediators of inflammatory responses, and appear to play a central role in the pathogenesis of many chronic inflammatory diseases (40, 41). It is now well that their biological activities in vivo are sufficient to reproduce local inflammation and matrix catabolism (42), by attracting and activating white blood cells to tissues, and stimulating their secretion of other lymphocytotropic cytokines and catabolic enzymes. Higher production of these cytokines have also been associated with response to infection, where local induction of IL-1 and TNF facilitates the elimination of the microbial invasion. Classic studies however also report that in some infectious conditions very high levels of monocytic cytokines are produced, which activate a cascade of concomitant events such as tissue catabolism, vascular reactivity and hyper-coagulation with damaging effects on the host (43, 44).
It has been demonstrated that there are stable inter-individual differences in the rates of production of IL-1 and TNF (45), and the ability to produce higher or lower cytokine levels clusters in families (46). More recently, particular gene variants have been associated with stable differences in IL-1 protein production in vitro from monocytes (47, 48), or TNF rate of transcription in transfix B cells (49). From these and other data, it is clear that genetic factors are playing a role in the IL-1 and TNF systems, and that IL-1 and TNF themselves are reasonable candidate genes for susceptibility or severity of various diseases that involve IL-1, TNF, or the mediators that they activate.
In a normal situation, the extent and severity of inflammation is regulated by feedback mechanisms to a level sufficient to fight microbial invasion without long-lasting damage to the tissues involved. It can be speculated that dysregulated production of IL-1 and/or TNF in some individuals would over-ride the feedback mechanisms and lead to tissue damage, matrix catabolism, and the activation of various processes that respond to certain levels of these mediators.
The IL-1 family is composed of at least six proteins produced by three genes. IL-1xcex1 and IL-1xcex2 are produced as propeptides of 31-33 KDa that are cleaved at the cell-membrane to 17 KDa mature proteins (50). Of the precursor proteins, only pro-IL-1 is biologically active. IL-1 receptor antagonist (IL-1 RA or IL-1RN) (51, 52) is produced either as a secretory peptide with a leader sequence or as an intracellular form based on alternative first exons. Both IL-1 RA proteins bind to the IL-1 receptor but have no agonist activity. The IL-1A, IL-1B and IL1RN genes all lie within a 430 kb region on the long arm of human chromosome 2 (53).
Transcription of IL-1 is activated in human monocytes by bacterial agents and other cytokines (IL-1 itself; IFNxcex3; IL-2; TNFxcex1). IL-1 agonists (IL-1xcex1 and IL-1xcex2) induce transcription of inducible cyclooxygenase (COX-2), nitric oxide synthetase, collagenase and other matrix metalloproteinases, as well as many cytokines (such as IL-2, IL-4, IL-6, IL-8 and TNFxcex1) (40). Down regulation of IL-1, up regulation of the IL-1 receptor, release of the soluble type II receptor, or predominance of IL-1RA, all limit the actions of IL-1, underlying the self-limitation of acute inflammation.
Similar biological actions are a consequence of tumour necrosis factor (TNF xcex1 or cachectin) or lymphotoxin (TNFxcex2 or LTxcex1) interaction with their receptor. TNFxcex1 is mainly produced by monocytes, and its biological functions are bard to separate from IL-1""s (41). Both TNFxcex1 and LT proteins are, however, products of separate genes, which lie in the Class III region of Chromosome 6, not distant from the MHC complex (54).
The functional correlates of these gene variants include protein dimorphism for IL-1A(+4845) (Ser for Ala at 114) (55) and direct association with levels of IL-1xcex2 protein production in vitro for IL-1B(+3954). In the TNF cluster, at least five microsatellites and five single base variations have been described (TABLE 1), and TNF(+308) has been associated with 8-fold higher transcriptional activation rate in vitro (49).
The early detection of a predisposition to genetic diseases presents the best opportunity for medical intervention in the progress of disease. Early prediction of risk may improve the prognosis for a patient through supervision and early intervention before the clinically detectable disorder occurs. In cases where patients with similar symptoms are treated with variable success, sophisticated genetic testing can differentiate individual patients with subtle or undetectable differences and can lead to more suitable individual treatments. Early intervention may involve methods such as gene therapy or treatment with drugs.
Genetic testing (also called genetic screening or genotyping) can be defined broadly as the testing of nucleic acid in an analytical capacity to determine if a patient has mutations (or alleles or polymorphisms) that either cause or increase susceptibility to a disease state or are in xe2x80x9clinkage disequilibriumxe2x80x9d with the gene causing a disease state.
With the development of genetic testing, it is now possible to identify gene mutations which indicate a propensity to develop disease, even when the disease is of polygenic origin. The number of diseases that can be identified by molecular biological methods continues to grow with increased understanding of the genetic basis of multifactorial disorders (see e.g., U.S. Pat. Nos. 4,582,788; 5,110,920; 4,801,531; 4,666,828; and 5,268,267). Genetic testing provides a means by which therapies can be targeted to those individuals in which they will be most effective depending upon the individuals genome type, a practice otherwise known as pharmacogenomics.
The IL-1 gene cluster is located on the long arm of chromosome 2 (2q13) and contains at least the genes for IL-1xcex1(IL-1A), IL-1xcex2(IL-1B), and the IL-1RN within a region of 430 Kb (Nicklin, et al., Genomics 19: 382-4 (1994)). The agonist molecules, IL-1xcex1 and IL-1xcex2, have potent pro-inflammatory activity and are at the head of many inflammatory cascades. Their actions, often via the induction of other cytokines such as IL-6 and IL-8, lead to activation and recruitment of leukocytes into damaged tissue, local production of vasoactive agents, fever response in the brain and the hepatic acute phase response. All three IL-1 proteins bind to type I and to type II IL-1 receptors, but only the type I receptor transduces a signal to the interior of the cell. In contrast, the type II receptor is shed from the cell membrane and acts as a decoy receptor. The receptor antagonist and the type II receptor, therefore, are both anti-inflammatory in their actions.
Inappropriate production of IL-1-axis components appears to play a central role in the pathology of many autoimmune and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disorder, psoriasis, and others. In addition, there are stable inter-individual differences in the rates of production of IL-1-axis components, and some of this variation may be accounted for by genetic differences at IL-1-axis gene loci (Molvig, et al., Scand. J. Immunol. 27:705-16 (1988); Pociot, et al., Eur. J. Clin. Invest. 22: 396-402 (1992)). Thus, the IL-1-axis genes are reasonable candidates for determining part of the genetic susceptibility to inflammatory diseases, most of which have a multifactorial etiology with a polygenic component.
Certain alleles from the IL-1 gene cluster are already known to be associated with particular disease states. For example, we have shown that IL-1RN allele 2 is associated with coronary artery disease (U.S. application Ser. No. 08/813,416), osteoporosis (U.S. Pat. No. 5,698,399, incorporated by reference herein), nephropathy in diabetes mellitus (Blakemore, et al., Hum. Genet. 97(3): 369-74 (1996)), alopecia areata (Cork, et al., J. Invest. Dermatol. 104(5 Supp.): 15S-16S (1995)), Graves disease (Blakemore, et al., J. Clin. Endocrinol. 80(1): 111-5 (1995)), systemic lupus erythematosus (Blakemore, et al., Arthritis Rheum. 37: 1380-85 (1994)), lichen sclerosis (Clay, et al., Hum. Genet. 94: 407-10 (1994)), and ulcerative colitis (Mansfield, et al., Gastoenterol. 106(3): 637-42 (1994)).
Likewise, the IL-1A allele 2 from marker xe2x88x92889 and IL-1B(TaqI) allele 2 from marker +3954 are associated with periodontal disease (U.S. Pat. No. 5,686,246, incorporated by reference herein). The IL-1A allele 2 from marker xe2x88x92889 is also associated with juvenile chronic arthritis, particularly chronic iridocyclitis (McDowell, et al., Arthritis Rheum. 38: 221-28 (1995)). The IL-1B(TaqI) allele 2 from marker +3954 of IL-1B is also associated with psoriasis and insulin dependent diabetes in DR3/4 patients (di Giovine, et al., Cytokine 7: 606 (1995); Pociot, et al., Eur J. Clin. Invest. 22: 396-402(1992)). Finally, the IL-1RN allele 1 is associated with diabetic retinopathy (GB Application No. 9618960.0).
Additionally, the following alleles from the IL-1 (33221461) haplotype are in linkage disequilibrium (GB Patent Application No. 9711040.7):
allele 3 of the 222/223 marker of IL-1A;
allele 3 of the gz5/gz6 marker of IL-1A;
allele 2 of the xe2x88x92889 marker of IL-1A;
allele 2 of the +3954 marker of IL-1B;
allele 1 of the xe2x88x92511 marker of IL-1B;
allele 4 of the gaat.p33330 marker;
allele 6 of the Y31 marker; and
allele 1 of the VNTR marker of IL-1RN.
Therefore, all of these alleles are associated to some degree with certain disease phenotypes.
However, although all of these alleles are in linkage disequilibrium with the actual disease-causing allele, none were previously believed to contribute to the disease state directly. Until now, no one has discovered an allele that produces a measurable phenotype which may actually contribute to the disease state.
The TNF locus in the class III region of the MHC is also a good candidate gene in autoimmune and inflammatory diseases, but because of the high degree of linkage disequilibrium across the MHC, it is difficult to determine which genes on a haplotype are important in the etiology of a disease. The haplotype HLA-A1-B8-DR3-DQ2, known as the autoimmune haplotype is associated with a number of autoimmune disease, including insulin dependent diabetes, Graves disease, myasthenia gravis, SLE, dermatitis herpetiformis and coeliac disease (61, 62, 63). A biallelic polymorphism at position xe2x88x92308 of the TNF promoter has been studied in these diseases, since it has been shown that (a) high TNF production levels have been associated with particular DR3 and DR4 haplotypes (46) and (b) that the TNF2 allele at xe2x88x92308 is carried on the autoimmune haplotype (64). However, in all the diseases mentioned above, it has not been possible to demonstrate any association of TNF with disease independently of the association with the autoimmune haplotype.
It seems that TNF does have an important role to play in infectious diseases; in a large study of patients with malaria in the Gambia, TNFxcex1 homozygosity was strongly associated with death from cerebral malaria, and no association with clinical outcome was found with any other marker in the class I and II regions of the MHC (65). Similar data have recently been reported in cutaneous leishmaniasis. (66).
During normal pregnancy, maternal hormones and locally-acting cytokines play a key role in regulating the onset of labor, cervical ripening, uterine contraction and delivery. Maternal infections during pregnancy have been demonstrated to perturb this normal cytokine and hormone regulated gestation, sometimes resulting in SPB. Recent findings have suggested that chronic infections that are not associated with the genitourinary tract may also contribute to SPB. Specifically, data suggest that a relatively common chronic oral infection, periodontitis, may provide sufficient challenge to the mother to trigger SPB. Data from pregnant animal models have demonstrated that low-grade, non-disseminating infections with P. gingivalis can result in diminished fetal growth. Furthermore, the magnitude of the fetal growth inhibition is inversely related to the maternal production of PGE2 and TNFxcex1, mimicking previous findings in humans. Experimental periodontitis in the pregnant hamster induced a 20% decrease in fetal weight (P=0.002). Periodontal infection is also associated with a significant rise in intra-amniotic PGE2 from 3.31xc2x11.1 ng/mL to 13.5xc2x14.1 ng/mL at P=0.03 in the hamster. Three independent case-control studies in humans have been conducted examining the relationship between periodontal status and SPB, suggesting that the periodontal status is worse in SPB mothers, as compared to normal birth weight, full-term delivery (FT) controls. In the first study of 48 mothers, periodontal status was significantly worse in cases vs controls (mean attachment levels 3.25xc2x10.05 mm vs 3.06xc2x10.06 mm, P=0.02). In a recently reported case-control study of 124 pregnant mothers, periodontal disease status was significantly worse in SPB mothers compared to FT controls (P less than 0.005), controlling for a variety of well-established risk factors. Findings from this study suggest that among primiparous mothers, the odds of being in the SPB group was elevated more than 7 fold if more severe periodontal disease was present. This association was significant even adjusting for a variety of obstetric confounders and covariates by logistic regression analyses. These findings of an association between periodontal disease and SPB have been supplemented with new data from a second 48 case-control study which have added more sensitive measures of current periodontal disease status. Results indicate that gingival crevicular fluid PGE2 (GCF-PGE2) levels are significantly higher in SPB mothers compared with FT controls [131.4xc2x121.8 vs 62.6xc2x110.3 (meanxc2x1SE, ng/mL), respectively at P=0.02]. Furthermore, within SPB mothers there was a significant inverse association between birth weight (as well as gestational age) and GCF-PGE2 levels at P=0.023. These data suggest a dose-response relationship for increasing GCF-PGE2, as a marker of current periodontal disease activity and decreasing birth weight. Furthermore, GCF-IL-1 levels were greatly elevated in SPB women, as compared to FT mothers, but the wide variance prevented statistical significance with this sample size [1217.8xc2x1281.3 ng/mL for SPB vs 720xc2x1105.2 ng/mL for FT]. In this third study the periodontal disease was more severe in PLBW mothers, as determined by biochemical and microbial biomarkers, but the difference in clinical attachment levels did not reach statistical significance (P=0.11).
In one aspect, the present invention provides novel methods for identifying whether a patient or a fetus is predisposed to an adverse pregnancy outcome such as premature preterm low birth weight delivery (LBW). In one embodiment, the method comprises determining whether an LBW associated allele is present in a nucleic acid sample obtained from the subject or the fetus. In a preferred embodiment, the LBW associated allele is IL-1A (+4845) allele 2 and/or an IL-1 (xe2x88x92511) allele 2, or alternatively a nucleic acid sequence that is in linkage disequilibrium with IL-1A (+4845) allele 2 and/or an IL-1 (xe2x88x92511) allele 2.
The LBW associated allele can be detected by any of a variety of techniques including: 1) performing a hybridization reaction between a nucleic acid sample and a probe that is capable of hybridizing to an LBW associated allele; 2) sequencing at least a portion of an LBW associated allele; or 3) determining the electrophoretic mobility of an LBW associated allele or fragment thereof (e g., fragments generated by endonuclease digestion). The allele can optionally be subjected to an amplification step prior to performance of the detection step. Preferred amplification steps are selected from the group consisting of: the polymerase chain reaction (PCR), the ligase chain reaction (LCR), stand displacement amplification (SDA), cloning, and variations of the above (e.g. RT-PCR and allele specific amplification). Primers for amplification may be selected to either flank the marker of interest (as required for PCR amplification) or directly overlap the marker (as in ASO hybridization). Oligonucleotides primers that hybridize to IL-1 and TNFA genes can easily be selected with commercially available primer selection programs. In a particularly preferred embodiment, the sample is hybridized with a set of primers, which hybridize 5xe2x80x2 and/or 3xe2x80x2 in a sense or antisense sequence to the ILD associated allele, and is subjected to a PCR amplification.
In another aspect, the invention features kits for performing the above-described assays. The kit can include nucleic acid sample collection means and a means for determining whether a subject carries an LBW associated allele. The kit may also comprise control samples, either negative or positive, or standards. The kit may also include an algorithmic device for assessing identity match. The algorithmic device may be used in conjunction with controls, or may be used independently of controls. The kits of the invention may also contain a variety of additional components such as a DNA amplification reagent, a polymerase, a nucleic acid purification reagent, a restriction enzyme, a restriction enzyme buffer, a nucleic acid sampling device, deoxynucleotides (dNTPs), and the like. Information obtained using the assays and kits described herein (alone or in conjunction with information on another genetic defect or environmental factor, which contributes to LBW) is useful for determining whether a pregnant, non-pregnant or non-symptomatic subject has or is likely to have a LBW baby, or more generally, a disease or condition that is caused by or contributed to by the allelic pattern detected. In addition, the information alone or in conjunction with information on another genetic defect contributing to LBW allows customization of thy for preventing the onset of symptoms associated with LBW, or for preventing the progression of the disease to end-stage, irreversible fibrosis. For example, this information can enable a clinician to: 1) more effectively prescribe a therapeutic that will address the molecular basis of LBW; and 2) better determine the appropriate dosage of a particular therapeutic for a particular subject.
In yet a further aspect, the invention features methods for treating or preventing the adverse pregnancy outcome of a low birth weight delivery in a subject, by administering to the subject, a pharmaceutically effective amount of an LBW therapeutic of the invention. In still another aspect, the invention provides in vitro and in vivo assays for screening test compounds to identify LBW therapeutics. In one embodiment, the screening assay comprises contacting a cell transfected with an LBW causative mutation that is operably linked to an appropriate promoter with a test compound and determining the level of expression of a protein in the cell in the presence and in the absence of the test compound. In a preferred embodiment, the LBW causative mutation results in decreased production of IL-1 receptor antagonist, and increased production of the IL-1 receptor antagonist or TNF-xcex1 in the presence of the test compound indicates that the compound is an agonist of IL-1 receptor antagonist or TNF-xcex1 activity. In another embodiment, the invention features transgenic non-human animals and their use in identifying antagonists of IL-1xcex1, IL-1xcex2 or TNF-xcex1 activity or agonists of IL-1Ra activity.
Other features and advantages of the invention will be apparent from the following detailed description and claims.